阅读说明：本技术 果糖在制备治疗缺血性损伤药物中的应用 (Application of fructose in preparation of medicine for treating ischemic injury ) 是由 周炳 梁静 吉训明 于盼盼 韩荣荣 于 2021-01-04 设计创作，主要内容包括：本发明公开了果糖在制备治疗缺血性脑卒中药物中的应用,涉及生物医药技术领域。包括以下方面：建立缺血损伤细胞模型及动物模型,对损伤模型进行果糖处理,发现果糖可调节神经元能量代谢与氧化还原代谢,减轻模型细胞与模型动物的缺血性损伤,具体为：降低缺血损伤后神经元死亡率,减少缺血损伤动物的脑梗死面积,并改善其神经行为学特征。(The invention discloses an application of fructose in preparation of a medicine for treating cerebral arterial thrombosis, and relates to the technical field of biological medicines. The method comprises the following steps: establishing an ischemia damaged cell model and an animal model, carrying out fructose treatment on the damaged model, finding that fructose can regulate neuron energy metabolism and redox metabolism, and relieving the ischemic damage of model cells and the model animal, specifically: reducing neuronal mortality after ischemic injury, reducing cerebral infarction area of animals with ischemic injury, and improving neurobehavioral characteristics of the animals.)

1. The application of fructose in preparing medicine for treating ischemic brain injury is provided.

2. Use according to claim 1, characterized in that the ischemic brain injury is ischemic stroke.

3. Use according to claim 2, characterized in that the fructosyl brain protective effect comprises: regulating energy metabolism and redox metabolism, increasing the survival rate of ischemia damaged neurons, reducing cerebral infarction area caused by ischemic stroke, and improving the neurobehavioral characteristics of ischemic stroke.

4. The use according to claims 1-3, wherein the route of administration of the medicament comprises intravenous injection, intramuscular injection, intraperitoneal injection, oral administration, nasal drop administration, sublingual administration, intracranial injection, interventional administration, implant administration, patch administration, or paint administration.

5. The use according to claims 1-3, wherein the medicament is in the form of injection, patch, drop, granule, tablet, effervescent tablet, pill, liniment, drop pill, sublingual tablet, microneedle patch, cream.

6. The use according to claims 1-3, wherein the medicament comprises a pharmaceutically acceptable pharmaceutical excipient.

7. The use according to claims 1-4, wherein the medicament is administered during a phase selected from the group consisting of cerebral infarction, surgery or drug therapy, stroke rehabilitation, pre-and post-thrombolytic.

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of fructose in preparation of a medicine for treating ischemic brain injury.

Background

Stroke is one of the most major diseases causing death and disability, and the burden on the society and families is severe. Ischemic stroke accounts for about 80% of patients with stroke. In the past years, the diagnosis and treatment of ischemic stroke have been greatly advanced, and most patients can be treated by medicines or operations. However, depending on thrombolytic and thrombolytic treatments within the time window, only about 46% of patients have good prognosis (90 days mRS0-2 min), and have risks of symptomatic bleeding and the like, and the comprehensive treatment effect is far lower than expected. Therefore, a more effective and safe strategy for preventing and treating cerebral arterial thrombosis is urgently needed.

When cerebral ischemic stroke occurs, local arterial vessel occlusion, cerebral blood flow reduction, deep hypoxia of brain tissues and microvascular dysfunction cause metabolic homeostasis imbalance. The blood supply, oxygen supply, sugar supply and the like of brain tissues are reduced or completely stopped, the redox metabolism is unbalanced, a large amount of oxygen free radicals are generated, and meanwhile, when the CBF is less than 10ml/(100 g-min), the energy of a penumbra is exhausted, cells are subjected to irreversible necrosis, and the cells become infarction cores. Therefore, when ischemia occurs, the maintenance of redox and energy metabolism steady state is crucial to relieving ischemia injury and promoting functional prognosis, and is an important ischemic stroke drug treatment target.

In the prior art, 1, 6-fructose diphosphate has the effect of regulating a plurality of enzyme activities in glucose metabolism because fructose diphosphate sodium is an important product of cell metabolism, exogenous fructose diphosphate sodium can act on cell membranes, and the concentrations of high-energy phosphate bonds and adenosine triphosphate in the cell membranes are increased by activating fructokinase on the cell membranes, so that potassium ion inflow is promoted to restore the resting state of the cells, the content of adenosine diphosphate in erythrocytes is increased, and the release of oxygen radicals and histamine is inhibited. Therefore, the compound can be used as a molecular level medicine for recovering and improving cell metabolism, and has the clinical auxiliary treatment effects on coronary heart disease, arrhythmia, acute cerebral infarction and other diseases. In the prior art, glycerol fructose injection is a high-permeability dehydration medicament. Wherein glycerol can participate in brain metabolism process, and improve brain metabolism; fructose can be metabolized without insulin; sodium chloride can adjust the electrolyte balance. The action mechanism of the product is as follows: after intravenous injection, the blood plasma osmotic pressure can be increased, so that water in tissues (including eyes, brain, cerebrospinal fluid and the like) enters blood vessels, thereby relieving tissue edema, and reducing intracranial pressure, intraocular pressure, cerebrospinal fluid volume and pressure thereof; the medicine can dilute blood, reduce edema around capillary, improve microcirculation, raise cerebral perfusion pressure, increase cerebral blood flow, and is one first-line medicine for lowering intracranial pressure and eliminating cerebral edema.

The common metabolic pathways of sugars in the body include glycolytic pathway (glycolytic pathway), pentose phosphate pathway (pentose phosphate pathway), and the like. The former is also called EMP pathway, which is a series of reactions that degrade glucose and glycogen into pyruvate with ATP production, and is a pathway of glucose degradation that is ubiquitous in all biological organisms. The glycolytic pathway, which is a common metabolic pathway for glucose to undergo aerobic or anaerobic decomposition, can occur under both anaerobic and aerobic conditions. The latter is a mode of oxidative decomposition of glucose. Since this pathway starts with glucose-6-phosphate (G-6-P), it is also called the hexose phosphate shunt. This pathway proceeds in the cytoplasm and can be divided into two stages. The first stage begins with the dehydrogenation of G-6-P to 6-phosphogluconolactone, followed by hydrolysis to 6-phosphogluconoacid and oxidative decarboxylation to 5-ribulose phosphate. NADP + is an electron acceptor in all the above oxidation reactions. In the second stage, the 5-ribulose phosphate is subjected to a series of ketone group conversion and aldehyde group conversion reactions, and finally generates glyceraldehyde-3-phosphate and fructose-6-phosphate through intermediate metabolites such as pentose phosphate, heptose phosphate and the like, and the two can enter the glycolysis pathway again for metabolism.

However, the fructose pathway has been studied in the prior art, and the fructose pathway has yet to be further explored for the redox and energy metabolism regulation of neurons, and there is no record of treating ischemic stroke through the fructose pathway. The invention reports that the fructose can regulate a metabolic network to treat cerebral ischemia injury for the first time.

Disclosure of Invention

The invention aims to provide application of fructose in preparation of a medicine for treating ischemic brain injury, wherein the fructose is used for adjusting energy metabolism and redox metabolism of neuronal cells so as to achieve the purpose of improving ischemic brain injury.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of fructose in preparing a pharmaceutical composition for treating cerebral ischemia injury. Fructose can improve ischemic injury, such as ischemic brain injury, by regulating neuronal energy metabolism and redox metabolism.

The fructose of the present invention can be administered by various administration routes including, but not limited to, intravenous administration, intramuscular injection, topical administration, intraperitoneal injection, intracranial injection, intrathoracic administration, intracranial administration, transpulmonary administration, subcutaneous administration, sublingual administration, oral administration, nasal drop administration, interventional administration, implant administration, patch administration, transdermal administration, film coating administration, or intrarectal administration, etc. Furthermore, the blood brain barrier effect is reduced in the forms of intravenous injection or intracranial administration and the like, and the medicine action efficiency is favorably improved.

The fructose can be administered before or after thrombolysis, in the acute cerebral infarction stage, the stroke rehabilitation stage.

The fructose can adopt different forms of pharmaceutical formulations, including but not limited to injections, powder injections, drops, patches, tablets, granules, sublingual tablets, micro-injections, effervescent tablets, solutions, emulsions, liposome preparations, suspensions, ointments, creams, transdermal absorbents, transmucosal absorbents, lozenges, drops, dripping pills, capsules, powders, liniments, fine granules, syrups and the like.

The pharmaceutical composition of the present invention further contains fructose or a pharmacologically acceptable salt thereof as an active ingredient, and if necessary, excipients, binders, disintegrants, lubricants, diluents, solubilizers, suspending agents, isotonic agents, pH adjusting agents, buffers, stabilizers, colorants, flavoring agents, flavors, and the like, which are generally used in the preparation of drugs, may be optionally added.

The pharmaceutical compositions of the present invention may be prepared, shaped or prepared according to methods generally used in the art, respectively. Further, it can be prepared into a state easy to store by freeze-drying, and it is used after being dissolved in a diluent such as water, physiological saline, buffer solution, etc. to prepare a suitable concentration.

The concentration of fructose in the pharmaceutical composition of the present invention may be 0.1-15mM, preferably 0.5-10mM, preferably 1-8mM, preferably 1-6 mM.

The ischemic injury of the invention comprises ischemic brain injury, ischemic stroke, and ischemic and anoxic injuries of other organs or parts, such as ischemic myocardial injury, ischemic kidney injury and the like.

The pharmaceutical composition of the present invention may be used alone or together with other active ingredients. The administration can be simultaneous or intermittent, and the intermittent administration can be within the time range that the effective components act simultaneously; the administration route and the administration method may be the same or different; the kit may be prepared separately or housed in a single package suitable for combined administration.

The invention has the beneficial effects that: according to the invention, a rat MCAO damage model and a neuron OGD damage model are utilized to observe the important role of a fructose passage in regulating neuron energy metabolism and redox metabolism for the first time, and a fructose passage metabolic map is drawn. The compound can inhibit the damage of ischemia to neurons, inhibit the behavioral change caused by the ischemia damage and has a protective effect on the cerebral ischemia damage. Meanwhile, the fructose is selected as an active ingredient, has an adjusting effect on the oxidation reduction and energy metabolism of ischemic brain injury neurons, can obviously improve the neuron activity, improve the activity and the survival rate of the injured neurons, improve neuroethology, reduce the cerebral infarction area, show a remarkable treatment effect on the ischemic brain injury, and is stronger than other similar medicines in the prior art.

Drawings

FIG. 1 shows metabolome changes in the cerebral cortex of a rat model of stroke;

FIG. 2 significant upregulation of the carbohydrate pathway following IPC treatment for 6 hours in rats;

FIG. 3 shows a fructose metabolic pathway temporal specificity regulation profile;

FIG. 4 shows the effect of fructose pathway on the ATP levels of DIV7 neurons;

FIG. 5 shows the effect of fructose pathway on DIV7 neuronal NADPH levels;

FIG. 6 shows the effect of fructose administration at different stages on survival of OGD-damaged neurons;

FIG. 7 shows the effect of fructose administration at different stages on the ATP production from OGD damaged neurons;

FIG. 8 shows the effect of fructose administration at different stages on the production of NADPH by OGD-damaged neurons;

FIG. 9 shows the distribution of isotopically labeled fructose in the PPP and TCA pathways under normal conditions and OGD treatment;

FIG. 10 shows the effect of treatment of different routes of administration of fructose on neuro-behavioral and cerebral infarction in MCAO rats;

FIG. 11 shows the effect of different concentrations of fructose on cell viability;

fig. 12 shows the effect of fructose, fructose-1, 6-diphosphate, glycerol fructose on the viability of DIV7 neurons, showing that fructose has a significant effect on enhancing neuronal viability;

FIG. 13 shows the effect of fructose, fructose-1, 6-diphosphate, glycerol fructose on the viability of OGD-damaged neurons, and the results show that fructose has a significant effect on reducing neuronal ischemic injury;

fig. 14 shows the effect of fructose, fructose-1, 6-diphosphate, and glycerol fructose on survival of OGD-injured neurons, and the results show that fructose has a significant effect on alleviating ischemic injury of neurons.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. The described embodiments are only some embodiments of the invention, not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: selection of fructose pathway

1.1 establishing ischemic cerebral apoplexy model

(1) Cell model: the method is characterized in that an oxygen sugar deprivation (OGD) model is utilized to simulate ischemic cerebral apoplexy, primary neurons cultured for 7 days (DIV7) in vitro are adopted, a culture solution is replaced by a glucose-free culture solution during treatment, the culture solution is placed in a hypoxia chamber (1% O2+ 5% CO2+ 94% N2), oxygen sugar deprivation is carried out for 1.5h at 37 ℃, a normal culture medium is replaced, and normal culture is continued for 24h under the normal oxygen condition.

(2) Animal model: an ischemic stroke is simulated by utilizing a middle arterial embolism induction (MCAO) injury model of a rat, and 250-300g of male SD rat is anesthetized by isopentane and fixed on an operating table in a supine manner. The neck skin is cut open, the subcutaneous tissue and the muscle are separated bluntly, the common carotid artery, the internal carotid artery and the external carotid artery are exposed, the common carotid artery is clamped by an artery clamp, and the internal carotid artery and the external carotid artery are respectively threaded with a ligature. Cutting a cut on an external carotid Artery, introducing a thread plug, slightly pushing the thread plug to the initial part of a Middle Cerebral Artery to form Middle Cerebral Artery blood supply interruption (MCAO for short), fixing a suture, drawing out the plug after 1.5h of operation, performing blood flow reperfusion, returning the animal to a cage for feeding for 24h, then performing neuroethology evaluation, killing after anesthesia, taking out the brain, performing TTC (time-to-live) staining, and observing the Cerebral infarction area.

1.2 ischemic stroke-induced metabolic disorders

We simulated the occurrence of ischemic stroke by constructing a cell model and a rat middle artery occlusion (MCAO) model, and studied the level change of metabolites in the brain cortex of a model rat. When ischemic stroke occurs, arterial vessel occlusion causes Cerebral Blood Flow (CBF) around the infarct core to be reduced, deep hypoxia of brain tissues and microvascular dysfunction, which causes metabolic homeostasis imbalance. The results show that the metabolic characteristics of the model group and the control group are significantly different (as shown in fig. 1A); the energy substrate NAD and reducing equivalent GSH, GSSG, NADPH are reduced (shown in figure 1B and figure 1C). After enriching the pathways of the differential metabolites, the TCA pathway, nicotinic acid and nicotinamide metabolic pathway, and GSH pathway were found to vary significantly (see fig. 1D), which are key pathways for maintaining energy and reducing equivalents homeostasis. The cerebral ischemic stroke is prompted to cause intracerebral metabolic disorder and is mainly reflected in the influence on energy and redox metabolism.

1.3 selection and determination of the fructose pathway

Ischemic stroke causes metabolic homeostasis imbalance, and an applicant team starts with the existing effective endogenous metabolic regulation and control means and searches for a target for treating ischemic stroke. Ischemic Preconditioning (IPC) is the most endogenous ischemic protection method, which initiates endogenous protection mechanisms by transient, non-lethal ischemic conditioning to make tissues tolerant to lethal ischemic injury.

Firstly, the rats are processed by IPC, the cerebral cortex is analyzed in metabolome at 0 hour, 2 hours, 6 hours and 24 hours after the IPC processing, the fact that the time specificity metabolic change (shown in figure 2) in the cerebral cortex of the rats is caused by the metabolite level after the IPC processing shows the time specificity, and after the IPC processing is carried out for 6 hours, the glycolysis channel, the PPP channel and the fructose channel are remarkably adjusted upwards, and the change is particularly remarkable. And the metabolites fructose-1-phosphate (F1P) and glycerol-3 phosphate (G3P) in the fructose pathway produced significant changes compared to the control group (as shown in FIG. 3). Suggesting that the fructose pathway is one of the important pathways of ischemic brain injury.

Secondly, to further verify the role of the fructose pathway in IPC ischemic protection, a fructose pathway inhibitor sorbini is added at a cellular level, and after the fructose pathway is found to be inhibited, the IPC does not enhance the ATP and NADPH contents any more (as shown in figures 4 and 5), thereby prompting the clear role of the fructose pathway in regulating ischemic brain injury, and the fructose pathway can participate in the maintenance of energy and reducing equivalent homeostasis.

Through target screening, a key component of a fructose channel, namely fructose, is finally selected, and the effect of the fructose on ischemic brain injury is verified at the animal and cell levels respectively.

Example 2: therapeutic effect of fructose on ischemic brain injury

2.1 evaluation index of ischemic brain injury

(1) Behavioral evaluation: and (3) performing behavioral evaluation by adopting a Longa scoring method, wherein the scoring standard is as follows: no nerve damage was scored 0, left forelimb stretch disorder 1, left circling 2, left leaning 3, unconscious 4, and dead 5.

(2) Determination of cerebral infarction area by TTC staining method: dissecting and separating out a rat whole brain, placing the rat whole brain in a mold, removing cerebellum and olfactory bulbs, cutting a coronal brain slice with the thickness of about 2mm, quickly placing the brain slice in 2% phosphoric acid buffer solution of triphenyltetrazolium chloride (TTC), incubating the rat whole brain slice in a dark place at 37 ℃ for 15-20 min, turning the rat whole brain slice once every 3-5 min in the incubating process, taking out the rat whole brain slice, cleaning the rat whole brain slice by PBS, soaking the rat whole brain slice in 4% PFA (paraformaldehyde) overnight, taking out the rat whole brain slice, cleaning the rat whole brain slice by PBS, taking a picture, and calculating the cerebral infarction area. And performing data statistics by using Graphpad Prism software, and comparing the means among the groups by using One-Way ANOVA (One-Way ANOVA), and comparing the means among the groups pairwise.

(3) Neuronal survival: TUNEL staining and immunofluorescence staining are adopted, neuron survival conditions are counted, neuron cell slide is carried out, 4% PFA is fixed for 10min, PBS is used for washing, incubation is carried out for 1h in a blocking buffer, MAP2 primary working solution is added, overnight at 4 ℃, PBS is used for washing, MAP2 secondary working solution is added, incubation is carried out for 1h at room temperature, after PBS is used for washing, TUNEL staining is carried out, and finally sealing agent containing DAPI is added for sealing. Data were collected by focusing microscope and the number of cell deaths was counted using ImageJ.

(4) ATP and NADPH content: separating cortical neurons by adopting a P0-P1 suckling mouse, adding a lentivirus transfection protein carrier for specifically detecting ATP and a lentivirus transfection protein carrier iNap for specifically detecting NADPH when inoculating the neurons, processing on the 7 th day, fixing by using 4% PFA, carrying out immunofluorescence staining, collecting data by using a confocal microscope, counting the fluorescence intensity of virus carrier protein by adopting ImageJ, and calculating the contents of ATP and NADPH.

2.2 Effect of fructose administration at different stages on OGD injured neurons

The energy metabolism and redox metabolism level of the neuron is characterized by detecting the expression quantity of a lentivirus transfection protein vector for specifically detecting ATP and a lentivirus transfection protein vector iNap for specifically detecting NADPH in a primary neuron culture system.

(1) Neuronal survival: DIV7 neurons were divided into four groups, control, OGD (with OGD treatment), OGD + Fru (per) with fructose treatment during OGD, and OGD + Fru (post) with fructose treatment during reperfusion. And (3) irrigating for 24h after OGD is finished, fixing by 4% PFA for 10min, washing by PBS, adding MAP2 primary anti working solution, washing by PBS, adding secondary anti working solution, washing by PBS, carrying out TUNEL dyeing, and finally adding a sealing agent containing DAPI for sealing. Neuronal survival was observed.

The results show that fructose added either during the OGD phase or during reperfusion reduces the rate of OGD-induced neuronal death, as shown in figure 6.

(2) ATP and NADPH content: cortical neurons were isolated from P0-P1 suckling mice, and at the same time as the neurons were inoculated, a lentiviral transfected protein vector for specifically detecting ATP and a lentiviral transfected protein vector iNap for specifically detecting NADPH were added, and on day 7, they were divided into four groups, namely, a control group, an OGD group (subjected to OGD treatment), an OGD + Fru (per) group (subjected to fructose treatment during OGD treatment), and an OGD + Fru (post) group (subjected to fructose treatment during reperfusion after OGD treatment). And (4) irrigating for 24h after the OGD is finished, fixing 4% PFA, carrying out MAP2 immunofluorescence staining, and observing the content of ATP and NADPH in neurons.

The results show that the addition of fructose in the OGD stage and the reperfusion stage can enhance the reduction of the ATP and NADPH production of neurons caused by OGD, namely the fructose can enhance the energy metabolism and the redox metabolism function of the neurons. As shown in fig. 7 and 8.

(3) Fructose increases influx into PPP and TCA pathways

By adopting a metabolic flux experiment, adding U-13C-fructose into a DIV7 neuron, respectively observing the distribution condition of the fructose in each metabolic pathway under normal conditions and after OGD treatment, finding that the labeling rate of key metabolite ribose-5-phosphate and erythrose-4-phosphate 13C on a PPP pathway is increased compared with that of a control group after OGD treatment, and indicating that the distribution of the fructose to the PPP pathway is enhanced when OGD occurs, namely the cell NADPH level is improved by enhancing the flow to the PPP so as to maintain the redox balance, and meanwhile, compared with the control group, the labeling rate of key metabolite oxaloacetate and citrate 13C on a TCA pathway is increased after OGD treatment. The results indicate that fructose influx into TCA is enhanced when OGD occurs, i.e. cellular energy supply is maintained by enhancing TCA. As shown in fig. 9.

2.3 Effect of fructose on MCAO rats after administration by different routes

Taking 250-charge 300g male SD rats, dividing into three groups, respectively carrying out MCAO molding treatment (MCAO group), MCAO molding and combined tail vein injection of fructose after thrombus extraction (MCAO + Fru-1 group), MCAO molding and combined abdominal cavity injection of fructose after thrombus extraction (MCAO + Fru-2 group), specifically carrying out the molding process of anaesthetizing rat isopentane, exposing right common carotid artery, internal carotid artery and external carotid artery, inserting a silica gel wire plug into the initial part of middle cerebral artery through external carotid artery, extracting the plug after 1.5h, immediately carrying out fructose administration according to the treatment scheme, carrying out behavioral evaluation by a Longa evaluation method after 24h administration, then carrying out anesthesia and death, taking out the brain, carrying out TTC staining and observing the cerebral infarction area.

The results show that the administration of fructose by two administration modes can improve the neurobehavioral characteristics of MCAO rats and reduce the cerebral infarction area. The tail vein injection effect is integrally superior to that of the abdominal cavity injection administration. As shown in fig. 10.

In conclusion, all experimental data show that fructose has obvious therapeutic action on OGD neurons and MCAO rats, and is expected to be developed into a novel medicament for treating ischemic cerebrovascular diseases, in particular to a novel medicament for treating ischemic stroke.

2.4 Effect of different concentrations of fructose on cell viability

By usingThe Luminescent cell viability test kit is used for researching the influence of fructose with different concentrations on the viability of neurons cultured for 7 days (DIV7), and the addition concentration of the fructose in a cell system is explored. The results show that the effect of increasing the activity of DIV7 neuronal cells is better when fructose is 1-6 mM. As shown in fig. 11.

Example 3: the effect of fructose, fructose-1, 6-diphosphate and glycerol fructose for preventing and treating ischemic injury is compared

Selecting the existing fructose-related medicaments in the prior art: fructose-1, 6-bisphosphate (F1,6P), glycerol fructose (g.fru.), the therapeutic effect on ischemic injury was verified by comparative experiments.

3.1 Effect of fructose, fructose-1, 6-bisphosphate, Glycerol fructose on the Activity of DIV7 neurons

By usingLuminescent cell viability test kit, the effect of different concentrations (0, 1, 2, 4, 6, 8, 16mM) of fructose (Fru.), fructose-1, 6-diphosphate (F1,6P), glycerol fructose (g.fru.) on neuronal viability in culture for 7 days (DIV7) was investigated.

The results show that the three can improve the neuron vitality, wherein the effect of the fructose on improving the neuron vitality is obviously better than that of the fructose-1, 6-diphosphate and glycerol fructose, as shown in figure 12 (compared with 0mM group, P is less than 0.05 for each concentration group).

3.2 protective Effect of fructose, fructose-1, 6-bisphosphate, Glycerol fructose on OGD (oxygen deprivation) injured neurons

By usingLuminecent cell viability test kit: culturing neuron cells by using a 96-well plate, performing OGD treatment on the neurons for 1.5 hours on the 7 th day, adding fructose, fructose-1, 6-diphosphate and glycerol fructose into the neurons respectively after the treatment, wherein the concentrations of the fructose, the fructose-1, 6-diphosphate and the glycerol fructose are all 2mM, and detecting the cell viability by using a kit after 24 hours.

The results show that OGD reduces cell viability remarkably, fructose can improve cell viability of OGD injured neurons remarkably, and fructose-1, 6-diphosphate and glycerol fructose have the tendency to improve viability of OGD injured neurons in the model system, but show significant difference. As shown in figure 13 (. P < 0.05,. P < 0.001).

Adopting an immunofluorescence staining method: after culturing neurons for 7 days, OGD treatment was performed for 1.5 hours, fructose-1, 6-diphosphate and glycerol fructose were added to the neurons at concentrations of 2mM, and MAP2 and DAPI staining were performed after 24 hours.

The results show that the OGD remarkably reduces the survival rate of neurons, the improvement effect of glycerol fructose on the survival rate of the neurons is slight after the drug treatment, and both fructose and fructose 1, 6-diphosphate have the effect of improving the survival rate of the neurons, wherein the effect of the fructose is remarkably superior to that of the fructose 1, 6-diphosphate. As shown in fig. 14 (× P < 0.05, × P < 0.01, × P < 0.001).